Partial Amplification and Sequencing of Gene Involved in Formation of Defensive White Line Reaction in Pseudomonas aeruginosa Using Degenerate Polymerase Chain Reaction

Pseudomonas lipopeptides such as white line-inducing principle (WLIP) have important antimicrobial activities. When a WLIP producer bacterium is grown close to P. tolaasii, a white line precipitate will be formed between them Identification of white line reaction (WLR) defense strategy and its distribution among different pseudomonads might help to use such an approach for the treatment of infectious diseases In current study, through a degenerate polymerase chain reaction (PCR) method, the partial characterization of genetic system of NRPS-based WLR was attempted in Pseudomonas aeruginosa for the first time in the world.
Domain analysis of non-ribosomal peptide synthetase (NRPS) enzymes was performed by the nonribosomal peptide synthetase-Polyketide synthases (NRPS-PKS) tool. Multiple DNA sequence alignments, phylogenetic analyses and local Blast searches were performed by Geneious Pro. The gDNA was extracted from P. aeruginosa LMG 1272 and degenerate PCR was carried out.
Findings: First, PCR amplification based on the C1 and TE domains was performed. Despite trying several modifications, a number of bands were obtained in addition to our expected band. For this purpose, the gene wlp/wip blast against Pseudomonas aeruginosa genomes was performed. Two homologes with about 50% identity to wlpB in RW10S2 were obtained. New primers were designed by which the target fragment could be amplified whose Blastp identified only one protein in bacterium Ralstonia solanacearum SD54 with the identity of about 43%.
This study shows that the defensive white line formation by P. aeruginosa is governed most likely by a genetic system different from what has been reported before.